Study On Establishing Method Of Sulfamethazine Determination

Veterinary drug resides has been a focus problem in food safety field.At present,the detection method of resides mainly includes biological effect determination, physico-chemical determination, HPLC,mass spectrometry,GC-MS,immunological determination,biosensor determination,ect. However, whether traditional conventional determination or standard accurate determination all universally exist some shortcomings,such as determined target being single,operating steps being miscellaneous,cost and instrument being expensive. Especially when simultaneously detecting various unknown pollutants,the shortcomings of lots of workload and time-consuming or force-dissipating ect appear undoubtedly.The detection technology need of fast,sensitive and accurately screening multiple unknown pollutants once also is increasing steadily.The establish of SM2 competitive inhibitory ELISA testing method The chicken simulated sample was tested by two ELISA method constructed,which recovery of testing sample was separately 98.607% and 85.152%; The regression equation and correlation coefficient of two testing method for SM2:Y=-26.728x+100.54, R2=0.9958 and Y=-28.049x+105.7.The linear range was separately 0.68~209ng/L and 1.52~621ng/L and The minimum detection limit for CAP was respectively 4.8ng/L and 5.3ng/L. The chicken simulated sample was tested by two ELISA method constructed,which recovery of testing sample was separately 99.341 % and 88.697%.The detection of SM2 HPLC With HPLC method, the regression equation of standard curve was y=0.0003x-3.799, correlation coefficient R2 being 0.9998, linear range of SM2 measuring being 0.049ng/mL~500ng/mL and the standard recovery average being 93.7%. When adding 0.5~0.125μg/g SM2 to muscle and hepatic sample of chicken, all of the average recovery of ELISA was higher than 85% and HPLC can confirm the detecting result of ELISA fairly.The cloning、synthesis、expression and activity assay of SM2 single chain variable fragment (scFv) antibody The heavy chain and light chain variable gene of SM2 monoclonal antibody was amplified by RT-PCR method from the total RNA of SM2 5D7 hybridona cell strain. Single chain variable fragment gene (VH-Linker-VL)was assembled by SOE-PCR method, which was cloned with pMD18-T vector. The sequence analysis showed that SM2-ScFv being 774bp encoded a deduced amino acid sequence of 258 residues,which 15 amino acids all were the flexible Linker connection between heavy chain and light chain. Gene sequence was analyzed by Igblast to get the variable gene of antibody light chain and heavy chain,which was in agreement with the characteristics of the mouse antibody variable region. Analysis of Amino Acids homology had the typical domains (IGV) of immunoglobulin and had high homology with single chain antibody registered,which was the mouse antibody variable gene sequence of the functional rearrangement.